Dealing with Pseudogenes in Molecular Diagnostics in the Next Generation Sequencing Era

Area of Science:

• Genomics
• Bioinformatics
• Clinical Genetics

Background:

• Pseudogenes pose significant challenges in next-generation sequencing (NGS) by contaminating genuine gene sequences.
• This contamination can lead to inaccurate variant detection, resulting in false positives and false negatives in clinical genetic analysis.

Purpose of the Study:

• To address the issues of pseudogene interference in NGS applications within a clinical context.
• To provide strategies for accurately analyzing genes with pseudogenes, focusing on target enrichment and mapping.

Main Methods:

• Discussion of PCR- and capture-based target enrichment strategies, including their pros and cons.
• Analysis of crucial parameters for mapping strategies to differentiate functional gene sequences from pseudogenic ones.
• Review of specific gene examples associated with Mendelian disorders and their NGS analysis approaches.

Main Results:

• The degree of pseudogene homology to the parent gene directly impacts variant detection accuracy.
• Effective target enrichment and mapping strategies are critical for mitigating pseudogene contamination.
• Various NGS approaches can successfully avoid pseudogene interference in clinical genetic analysis.

Conclusions:

• Accurate variant detection in genes with pseudogenes requires careful selection of NGS strategies.
• Implementing robust target enrichment and mapping protocols is essential for reliable clinical genetic testing.
• Understanding and overcoming pseudogene challenges enhances the utility of NGS for diagnosing genetic disorders.